Equilibrium binding of thrombin to recombinant human thrombomodulin: effect of hirudin, fibrinogen, factor Va, and peptide analogues

Thrombomodulin is an endothelial cell surface receptor for thrombin that acts as a physiological anticoagulant. The properties of recombinant human thrombomodulin were studied in COS-7, CHO, CV-1, and K562 cell lines. Thrombomodulin was expressed on the cell surface as shown by the acquisition of thrombin-dependent protein C activation. Like native thrombomodulin, recombinant thrombomodulin contained N-linked oligosaccharides, had Mr approximately 100,000, and was inhibited or immunoprecipitated by anti-thrombomodulin antibodies. Binding studies demonstrated that nonrecombinant thrombomodulin expressed by A549 carcinoma cells and recombinant thrombomodulin expressed by CV-1 and K562 cells had similar Kd's for thrombin of 1.3 nM, 3.3 nM, and 4.7 nM, respectively. The Kd for DIP-thrombin binding to recombinant thrombomodulin on CV-1(18A) cells was identical with that of thrombin. Increasing concentrations of hirudin or fibrinogen progressively inhibited the binding of 125I-DIP-thrombin, while factor Va did not inhibit binding. Three synthetic peptides were tested for ability to inhibit DIP-thrombin binding. Both the hirudin peptide Hir53-64 and the thrombomodulin fifth-EGF-domain peptide Tm426-444 displaced DIP-thrombin from thrombomodulin, but the factor V peptide FacV30-43 which is similar in composition and charge to Hir53-64 showed no binding inhibition. The data exclude the significant formation of a ternary complex consisting of thrombin, thrombomodulin, and hirudin. These studies are consistent with a model in which thrombomodulin, hirudin, and fibrinogen compete for binding to DIP-thrombin at the same site.     

Activation of human factor V by factor Xa and thrombin

The activation of human factor V by factor Xa and thrombin was studied by functional assessment of cofactor activity and sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by either autoradiography of 125I-labeled factor V activation products or Western blot analyses of unlabeled factor V activation products. Cofactor activity was measured by the ability of the factor V/Va peptides to support the activation of prothrombin. The factor Xa catalyzed cleavage of factor V was observed to be time, phospholipid, and calcium ion dependent, yielding a cofactor with activity equal to that of thrombin-activated factor V (factor Va). The cleavage pattern differed markedly from the one observed in the bovine system. The factor Xa activated factor V subunits expressing cofactor activity were isolated and found to consist of peptides of Mr 220,000 and 105,000. Although thrombin cleaved the Mr 220,000 peptide to yield peptides previously shown to be products of thrombin activation, cofactor activity did not increase. N-Terminal sequence analysis confirmed that both factor Xa and thrombin cleave factor V at the same bond to generate the Mr 220,000 peptide. The factor Xa dependent functional assessment of 125I-labeled factor V coupled with densitometric analyses of the cleavage products indicated that the cofactor activity of factor Xa activated factor V closely paralleled the appearance of the Mr 220,000 peptide. This observation facilitated the study of the kinetics of factor V activation by allowing the activation of factor V to be monitored by the appearance of the Mr 220,000 peptide (factor Xa activation) or the Mr 105,000 peptide (thrombin activation). Factor Xa catalyzed activation of factor V obeyed Michaelis-Menten kinetics and was characterized by a Km of 10.4 nM, a kcat of 2.6 min-1, and a catalytic efficiency (kcat/Km) of 4.14 X 10(6) M-1 s-1. The thrombin-catalyzed activation of factor V was characterized by a Km of 71.7 nM, a kcat of 14.0 min-1, and a catalytic efficiency of 3.26 X 10(6) M-1 s-1. This indicates that factor Xa is as efficient an enzyme toward factor V as thrombin.     

The action of factor Xa, thrombin and trypsin on human factor II

The proteolytic action of human and bovine Factor Xa, bovine thrombin and bovine pancreatic trypsin Factor II at pH 7.5 and 25°C was monitored by sodium dodecylsulfate gel electrophoresis and thrombin assays. Purified human and bovine Factor Xa, and trypsin, were found to activate Factor II to thrombin. The conversion of Factor II to thrombin by either Factor Xa or trypsin was found to proceed through two thrombogenic intermediates. The reaction pathway appears to be sequential in that the Factor II (75 000 daltons) is first cleaved to a 55 000-dalton thrombogenic product (Intermediate 1) and a 25 000-dalton non-thrombogenic product (Fragment 1). Intermediate 1 is subsequently converted to an inactive 37 000-dalton thrombogenic protein (Intermediate 2) and a 16 000-dalton protein (Fragment 2). Intermediate 2 is finally converted to an active 37 000-dalton thrombin (α-thrombin). Purified bovine thrombin readily converted Factor II to Intermediate 1 and Fragment 1, but possessed little capacity to catalyze subsequent cleavages to produce active thrombin. The ability of thrombin to cleave Factor II was entirely obviated in the presence of hirudin. Under the conditions of the incubation, the maximum thrombin yield obtainable by Factor Xa or trypsin activation was 50% when compared to the two-stage potential thrombin.     

From selective substrate analogue factor Xa inhibitors to dual inhibitors of thrombin and factor Xa. Part 3

Highly potent and selective substrate analogue factor Xa inhibitors were obtained by incorporation of non-basic or modestly basic P1 residues known from the development of thrombin inhibitors. The modification of the P2 and P3 amino acids strongly influenced the selectivity and provided potent dual factor Xa and thrombin inhibitors without affecting the fibrinolytic enzymes. Several inhibitors demonstrated excellent anticoagulant efficacy in standard clotting assays in human plasma.     

Macromolecular specificity determinants on thrombin for fibrinogen and thrombomodulin

The endothelial cell surface membrane protein thrombomodulin binds thrombin with high affinity and acts as both a cofactor for protein C activation and an inhibitor of fibrinogen hydrolysis. We have previously shown that bovine thrombomodulin is a competitive inhibitor of fibrinogen binding to thrombin but has no effect on thrombin activity toward tripeptide substrates or antithrombin III. Hence, thrombomodulin and fibrinogen may share macromolecular specificity sites on thrombin which are distinct from the active site. In this investigation, we have studied the interaction of thrombin-thrombomodulin with fibrinogen and various thrombin derivatives. We show that fibrinogen is a competitive inhibitor of thrombomodulin binding to thrombin, with a Kis = 10 microM. Thrombin derivatives (bovine (pyridoxal phosphate)4-thrombin and human thrombin Quick I), which bind fibrinogen with much reduced affinity, are shown to also interact with thrombomodulin with greatly reduced affinity. These results are consistent with the hypothesis that thrombomodulin and fibrinogen share macromolecular specificity sites on thrombin.     

New synthetic heparin mimetics able to inhibit thrombin and factor Xa.

Synthetic pentadeca-, heptadeca- and nonadecasaccharides, comprising an antithrombin III (AT III) binding pentasaccharide prolonged at the non-reducing end by a thrombin binding domain have been obtained. The pentadecasaccharide is the shortest oligosaccharide able to catalyse thrombin inhibition by AT III. The nonadecasaccharide is a more potent thrombin inhibitor than standard heparin.     

Further localization of binding sites for thrombin and protein C in human thrombomodulin

To elucidate the binding sites for thrombin and protein C in the six epidermal growth factor (EGF) domains of human thrombomodulin, recombinant mutant proteins were expressed in COS-1 cells. Mutant protein EGF456, which contains the fourth, fifth, and sixth EGF domains from the NH2 terminus of thrombomodulin, showed complete cofactor activity in thrombin-catalyzed protein C activation, as did intact thrombomodulin or elastase-digested thrombomodulin. EGF56, containing the fifth and sixth EGF domains, did not have cofactor activity; but EGF45, containing the fourth and fifth EGF domains, had about one-tenth of the cofactor activity of EGF456. Thrombin binding to attached recombinant thrombomodulin (D123) was inhibited by EGF45 as well as by EGF56. A synthetic peptide (ECPEGYILDDGFICTDIDE), corresponding to Glu-408 to Glu-426 in the fifth EGF domain, inhibited thrombin binding to attached thrombomodulin (D123) with an apparent Ki of 95 microM. At Ca2+ concentrations of 0.25-0.3 mM, intact protein C was maximally activated by thrombin in the presence of EGF45, EGF456, or EGF1-6, which contains the first to sixth EGF domains; but such maximum cofactor activity was not observed when gamma-carboxyglutamic acid-domainless protein C was used. These findings suggest that: 1) thrombin binds to the latter half of the fifth EGF domain; and 2) protein C binds to the fourth EGF domain of thrombomodulin through Ca2+ ions.     

Heparin enhances the specificity of antithrombin for thrombin and factor Xa independent of the reactive center loop sequence. Evidence for an exosite determinant of factor Xa specificity in heparin-activated antithrombin

Heparin activates the primary serpin inhibitor of blood clotting proteinases, antithrombin, both by an allosteric conformational change mechanism that specifically enhances factor Xa inactivation and by a ternary complex bridging mechanism that promotes the inactivation of thrombin and other target proteinases. To determine whether the factor Xa specificity of allosterically activated antithrombin is encoded in the reactive center loop sequence, we attempted to switch this specificity by mutating the P6-P3' proteinase binding sequence excluding P1-P1' to a more optimal thrombin recognition sequence. Evaluation of 12 such antithrombin variants showed that the thrombin specificity of the serpin allosterically activated by a heparin pentasaccharide could be enhanced as much as 55-fold by changing P3, P2, and P2' residues to a consensus thrombin recognition sequence. However, at most 9-fold of the enhanced thrombin specificity was due to allosteric activation, the remainder being realized without activation. Moreover, thrombin specificity enhancements were attenuated to at most 5-fold with a bridging heparin activator. Surprisingly, none of the reactive center loop mutations greatly affected the factor Xa specificity of the unactivated serpin or the several hundred-fold enhancement in factor Xa specificity due to activation by pentasaccharide or bridging heparins. Together, these results suggest that the specificity of both native and heparin-activated antithrombin for thrombin and factor Xa is only weakly dependent on the P6-P3' residues flanking the primary P1-P1' recognition site in the serpin-reactive center loop and that heparin enhances serpin specificity for both enzymes through secondary interaction sites outside the P6-P3' region, which involve a bridging site on heparin in the case of thrombin and a previously unrecognized exosite on antithrombin in the case of factor Xa.     

Determinants of the specificity of protease-activated receptors 1 and 2 signaling by factor Xa and thrombin

Factor Xa (FXa) elicits intracellular signaling responses through the activation of protease-activated receptor 2 (PAR2) and possibly also through PAR1 in endothelial cells. In this study, we investigated FXa signaling in endothelial cells when the protease was either in free form or assembled into the prothrombinase complex. Furthermore, we prepared several wild-type and mutant PAR1 and PAR2 cleavage-reporter constructs in which their exodomains were fused to cDNA encoding for a soluble alkaline phosphatase (ALP). In the mutants, P2 residues were exchanged between PAR1 and PAR2 cleavage-reporter constructs and the hirudin-like binding site (HLBS) of PAR1 was inserted into the homologous site of PAR2. In non-transfected cells, FXa elicited a protective response which could be blocked by a specific anti-PAR2 but not by an anti-PAR1 antibody. A similar protective activity was observed for FXa in the prothrombinase complex. Further studies revealed that neither the Gla- nor EGF1-domain of FXa is required for its signaling activity, however, the N-terminus Arg-86 and Lys-87 of the EGF2-domain were essential. In the cleavage-reporter transfected cells, FXa cleaved the PAR2 construct effectively, however, replacing its P2-Gly with P2-Pro of PAR1 impaired its cleavage by FXa but improved it by thrombin. A PAR2 construct containing both P2-Pro and HLBS of PAR1 was poorly cleaved by FXa, but effectively by thrombin. A PAR1 construct containing P2 and P3 residues of PAR2 was poorly cleaved by thrombin but effectively by FXa. These results provide new insight into mechanisms through which coagulation proteases specifically interact with their target PAR receptors.     

Functional characterization of human platelet-released factor V and its activation by factor Xa and thrombin

The functional characterization of human platelet-released factor V and its activation by factor Xa and thrombin was studied by functional assessment of cofactor activity and Western blotting analyses of platelet releasates, obtained by stimulating washed suspensions of platelets with various agonists, including collagen, collagen with ADP, and the calcium ionophore A23187. Platelet factor V was released as a partially proteolyzed molecule that was bound to platelet microparticles, irrespective of the agonist used. Radiolabeled plasma factor V was not cleaved for up to 30 min following release when added to platelets prior to stimulation, suggesting that platelet factor V was stored in a partially proteolyzed form. Released platelet factor V possessed significant cofactor activity that was increased only 2-3-fold by either factor Xa or thrombin. The factor V subunits that expressed cofactor activity were isolated and found to consist of peptides of Mr = 220,000 and 150,000. Incubation of released platelet factor V with factor Xa or thrombin yielded the same cleavage pattern, in which two peptides of Mr = 105,000 and 74,000 appeared to be electrophoretically indistinguishable from thrombin-activated plasma factor V. Under the conditions of these studies, factor Xa activated platelet-released factor V 50-100 times more effectively than thrombin. This observation may be due in part to the existence of platelet factor V in a partially proteolyzed state, or its association with platelet microparticles following platelet stimulation. These data collectively suggest that platelet-released factor V may be the foremost initiator of prothrombinase complex assembly and function during the early stages of coagulation with additional cofactor activation accomplished by factor Xa.     

Monoclonal antibodies for human thrombomodulin which recognize binding sites for thrombin and protein C

Monoclonal antibodies for human thrombomodulin, a cofactor for thrombin-catalyzed activation of protein C, were prepared and their epitopes characterized. All six antibodies (MFTM-1-MFTM-6) bound to an elastase-digested active fragment of thrombomodulin, which contains six consecutive EGF domains. Binding of thrombomodulin to these antibodies did not depend on Ca2+ concentration. MFTM-4, MFTM-5, and MFTM-6 strongly inhibited protein C activation by thrombin and thrombomodulin. MFTM-4 and MFTM-5 inhibited thrombin binding to fixed thrombomodulin and bound to a recombinant mutant EGF456 protein, which contained the fourth, fifth, and sixth EGF domains of thrombomodulin. However, MFTM-6 did not inhibit thrombin binding to thrombomodulin and did not bind to EGF456 protein. Binding of thrombomodulin to fixed MFTM-4 or MFTM-5 was competitively inhibited by a recombinant mutant EGF45 protein which contained the fifth and sixth EGF-domains. These results suggest that epitopes of MFTM-4 and MFTM-5 are located in the fifth EGF domain of thrombomodulin. Thus, the binding site for thrombin is located in the fifth EGF domain. These results also suggest that an epitope for MFTM-6 is located at a region near the binding site for gamma-carboxyglutamic acid residues of protein C via Ca2+ on thrombomodulin.     

Activation of duck prothrombin by factor Xa and thrombin

Prothrombin isolated from duck sodium citrate plasma was activated in a system containing duck factor Xa and calcium ions. Polyacrylamide gel electrophoresis showed that intermediates and the final product, thrombin, of Mr in the range 21 500-52 000 were present in the incubation mixture Serine and isoleucine were found to be the N-terminal amino acids of the intermediate form 1 and thrombin, respectively.     

The effect of bovine thrombomodulin on the specificity of bovine thrombin

Bovine lung thrombomodulin is purified and used to investigate the basis of the change in substrate specificity of bovine thrombin when bound to thrombomodulin. Bovine thrombomodulin is a single polypeptide having an apparent molecular weight of 84,000 and associates with thrombin with high affinity and rapid equilibrium, to act as a potent cofactor for protein C activation and antagonist of reactions of thrombin with fibrinogen, heparin cofactor 2, and hirudin. Bovine thrombomodulin inhibits the clotting activity of thrombin with Kd less than 2.5 nM. Kinetic analysis of the effect of bovine thrombomodulin on fibrinopeptide A hydrolysis by thrombin indicates competitive inhibition with Kis = 0.5 nM. The active site of thrombin is little perturbed by thrombomodulin, as tosyl-Gly-Pro-Arg-p-nitroanilide hydrolysis and inhibition by antithrombin III are unaffected. Insensitivity of the reaction with antithrombin III is likewise observed with thrombin bound to thrombomodulin on intact endothelium. Antithrombin III-heparin, human heparin cofactor 2, and hirudin inhibit thrombin-thrombomodulin more slowly than thrombin. These effects may arise from a decrease in Ki of the inhibitors for thrombin-thrombomodulin or from changes in the active site not detected by tosyl-Gly-Pro-Arg-p-nitroanilide or antithrombin III. Bovine prothrombin fragment 2 inhibits thrombin clotting activity (Kd less than 7.5 microM) and acts as a competitive inhibitor of protein C activation (Kis = 2.1 microM). The data are consistent with a mechanism whereby thrombomodulin alters thrombin specificity by either binding to or allosterically altering a site on thrombin distinct from the catalytic center required for binding or steric accommodation of fibrinogen, prothrombin fragment 2, heparin cofactor 2, and hirudin.     

Ligand binding specificity of the leukocyte response integrin expressed by human neutrophils.

The ligand binding specificity of the leukocyte response integrin (LRI) expressed by polymorphonuclear leukocyte (PMN) was investigated by examining its interaction with two adhesion motifs within fibrinogen: the alpha chain sequence RGD and the gamma chain sequence KQAGDV. The effect of the hexapeptides KQAGDV, KQRGDV, and KGAGDV on fibrinogen-stimulated phagocytosis, a LRI-dependent function, was examined. Surprisingly, the sequence KGAGDV was most potent for inhibition of fibrinogen-stimulated ingestion; the order of potency of these peptides was KGAGDV greater than KQAGDV greater KQRGDV = GRGDSPA. Latex spheres coated with multivalent KGAGDV bound specifically to PMN and antibodies that recognized either the LRI beta chain (7G2) or an associated protein (IAP)-abrogated bead binding. Various control and anti-beta 1 and anti-beta 2 antibodies did not affect bead binding. Monovalent peptides KGAGDV and KQRGDV were equipotent for inhibition of bead binding to unstimulated PMN (ID50 = 19 microM). In contrast, KGAGDV was more potent than KQRGDV for inhibition of bead binding to N-formylmethionylleucylphenylalanine-stimulated PMN (ID50 = 2.5 microM versus ID50 = 60 microM). A control peptide, KGALEVA, did not inhibit LRI ligand binding or function. These data suggest that the unique amino acid sequence KGAGDV may represent a specific ligand for LRI and that LRI ligand binding specificity may be regulated by the activation state of the cell.     

Substitution of asparagine for arginine 347 of recombinant factor Xa markedly reduces factor Va binding

Herein we describe a recombinant factor X (fX) with a single substitution at position 347 (fXR347N). Activated fXR347N had a reduced affinity for factor Va (fVa), although the catalytic impact of fVa binding remained intact. The mutation was selective as demonstrated by normal activation and inhibition, except in the presence of subsaturating heparin where the rate of inhibition by antithrombin III (ATIII) was 15% of normal. The reactivity of fXaR347N toward prothrombin was equivalent to wild-type fXa (fXaWT) in the absence of fVa and phospholipid. Addition (without phospholipid) of fVa dramatically increased the catalytic efficiency of fXaWT toward prothrombin but had a negligible effect on fXaR347N. On addition of phosphatidylcholine:phosphatidylserine (PC:PS, 3:1) vesicles, fXaR347Ndisplayed an increased catalytic activity in response to fVa, but the apparent affinity for fVa on the phospholipid surface was 5-20-fold lower than that of fXaWT. On an activated platelet surface, however, fXaWT and fXaR347N activated prothrombin similarly. In a competitive binding assay that measures the displacement of radiolabeled fXa from fVa on a phospholipid surface, fXaR347N was approximately 10-fold less effective than fXaWT. Substitution of fXa at position 347 selectively attenuates the interaction between fXa and fVa without affecting its catalytic activity.     

Role of regulatory exosite I in binding of thrombin to human factor V, factor Va, factor Va subunits, and activation fragments.

The blood coagulation proteinase, thrombin, converts factor V into factor Va through a multistep activation pathway that is regulated by interactions with thrombin exosites. Thrombin exosite interactions with human factor V and its activation products were quantitatively characterized in equilibrium binding studies based on fluorescence changes of thrombin covalently labeled with 2-anilinonaphthalene-6-sulfonic acid (ANS) linked to the catalytic site histidine residue by Nalpha-[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH2Cl ([ANS]FPR-thrombin). Exosite I was shown to play a predominant role in the binding of factor V and factor Va from the effect of the exosite I-specific ligand, hirudin54-65, on the interactions. Factor V and factor Va bound to exosite I of [ANS]FPR-thrombin with similar dissociation constants of 3.4 +/- 1.3 and 1.1 +/- 0.4 microM and fluorescence enhancements of 182 +/- 41 and 127 +/- 17%, respectively. Native thrombin and labeled thrombin bound with similar affinity to factor Va. Among factor V activation products, the factor Va heavy chain was shown to contain the site of exosite I binding, whereas exosite I-independent, lower affinity interactions were observed for activation fragments E and C1, and no detectable binding was observed for the factor Va light chain. The results support the conclusion that the factor V activation pathway is initiated by exosite I-mediated binding of thrombin to a site in the heavy chain region of factor V that facilitates the initial cleavage at Arg709 to generate the heavy chain of factor Va. The results further suggest that binding of thrombin through exosite I to factor V activation intermediates may regulate their conversion to factor Va and that similar binding of thrombin to the factor Va produced may reflect a mode of interaction involved in the regulation of prothrombin activation.     

Specificity of soluble phospholipid binding sites on human factor Xa

We explore here the specificities of lipid regulatory sites on factor X(a) that affect the rate of factor X(a)-catalyzed prothrombin activation. We examined a series of 11 phosphatidylserine (PS) analogues in order to map the structural features of a lipid molecule that are needed to elicit both the structural response and the full increase in activity that can be obtained with the PS molecule. Our observations are interpreted in terms of a model in which factor X(a) is regulated by sequential occupancy of a pair of linked lipid binding sites, each of which have different minimum ligand structural requirements to induce structural changes. The first site is apparently of higher affinity and recognizes diacylglycerol (DAG) as a minimal binding structure. The second site is occupied with an affinity slightly less than the first site only when the first is occupied, but binds PS with very low affinity otherwise. It recognizes glycerophosphorylserine (GPS) as the minimal ligand. To test this interpretation, experiments were performed in which more than one lipid species was present. It was necessary to invoke the existence of factor X(a) species containing different lipids at each site, each having different structural and functional responses. For optimal activity enhancement, both binding sites must be occupied, the first by PS, although the second can be occupied with other lipids.     

Are factor Xa inhibitors superior to thrombin inhibitors in anticoagulation?

Protease inhibitors are useful tools for increasing the inhibitor potential of plasma. In this context, thrombin inhibitors attracted special interest. However, other clotting enzymes, especially factor Xa, are target enzymes of protease inhibitors besides thrombin. Our studies on structure-activity relationships of benzamidine derivatives resulted in selective inhibitors of thrombin and factor Xa. The use of these inhibitors enabled us to clarify whether the antithrombin activity or the anti-factor Xa activity of a compound is more efficient in anticoagulation. We assessed the concentration-dependent inhibition of the activated partial thromboplastin time by these compounds. If one correlates the inhibitor concentration, which prolonged the clotting time by 60 s, with the dissociation constants one will realize that thrombin inhibition is significantly more efficient in anticoagulation than inhibition of factor Xa.     

Role of factor VIII C2 domain in factor VIII binding to factor Xa.

Factor VIII (FVIII) is activated by proteolytic cleavages with thrombin and factor Xa (FXa) in the intrinsic blood coagulation pathway. The anti-C2 monoclonal antibody ESH8, which recognizes residues 2248-2285 and does not inhibit FVIII binding to von Willebrand factor or phospholipid, inhibited FVIII activation by FXa in a clotting assay. Furthermore, analysis by SDS-polyacrylamide gel electrophoresis showed that ESH8 inhibited FXa cleavage in the presence or absence of phospholipid. The light chain (LCh) fragments (both 80 and 72 kDa) and the recombinant C2 domain dose-dependently bound to immobilized anhydro-FXa, a catalytically inactive derivative of FXa in which dehydroalanine replaces the active-site serine. The affinity (K(d)) values for the 80- and 72-kDa LCh fragments and the C2 domain were 55, 51, and 560 nM, respectively. The heavy chain of FVIII did not bind to anhydro-FXa. Similarly, competitive assays using overlapping synthetic peptides corresponding to ESH8 epitopes (residues 2248-2285) demonstrated that a peptide designated EP-2 (residues 2253-2270; TSMYVKEFLISSSQDGHQ) inhibited the binding of the C2 domain or the 72-kDa LCh to anhydro-FXa by more than 95 and 84%, respectively. Our results provide the first evidence for a direct role of the C2 domain in the association between FVIII and FXa.